With the accelerated development and use of nucleic acid microarray technology, there is considerable interest in applying existing (off-the-shelf) microarray methods and devices in uncharacterized sample backgrounds, and developing microfluidic devices for near-instantaneous biodetection applications. Uncharacterized sample backgrounds create both a sample preparation and data interpolation challenge for diagnostic use of microarray technology, and a significant engineering challenge for packaging microarray processes within a sample-to-answer fluidic test cartridge. The challenges become even more acute within the context of environmental monitoring due to the co-extraction of soluble environmental constituents that interfere with molecular techniques (including PCR amplification, hybridization, and fluorescent detection) and the preponderance of unknown and uncharacterized non-target organisms in the biological background.
Theoretical and experimental data with planar and gel element arrays show that mismatched targets preferentially bind under non-equilibrium hybridization conditions, exacerbating the problem of false positive detection. Depending upon the nucleic acid purification and labeling strategy, non-target sequences can also contribute to increased local and global background, degrading overall system (sample-to-answer) performance and dynamic range. One strategy to address cross-hybridization in defined (or closed) biological systems is to remove unpredictable probes from the array. Another is to increase the total number of probes on an array and statistically compare the signal intensity between perfectly matched (PM) and single base mismatched (MM) duplexes, typifying re-sequencing array designs. Hybridization kinetics can also be used to de-convolve false-positives in defined biological systems, and temperature, ionic strength and chemical additives are well-known methods of influencing hybridization stringency. However, a question concerning the application of microarray technology in uncharacterized samples or open biological systems is: how is it possible to know if and when hybridization signals result from a perfectly matched or mismatched probe: target combination? In an uncharacterized sample, any detectable microarray signal (over background) may have practical importance (e.g. pathogen surveillance), and recent work indicates how PM and MM probe comparisons can be problematic (e.g. erroneous) in complex samples.
One approach to de-convolve false positive hybridizations on microarray substrates has been to generate post-hybridization thermal dissociation curves for every probe on the array. Historically, dissociation studies were aimed at understanding nucleotide mismatch discrimination, duplex stability and hybridization behavior in order to define an a priori hybridization conditions for generating unambiguous reads from the initial hybridization data Another way to utilize on-chip thermal dissociation, however, is as a post-hybridization, diagnostic indicator of hybridization specificity, utilizing curve shape and/or dissociation constants as part of the decision logic for data interpretation, being careful to account for thermal effects on commonly used fluorescent reporters.
From a sensor or biodetection technology perspective, post-hybridization thermal dissociation analysis is an exciting possibility for target-independent, diagnostic validation of hybridization specificity irrespective of a priori knowledge of target background. By itself, however, the technique does little to address the (fluidic or automated) nucleic acid sample preparation challenge or simplify the attendant analysis instrumentation.